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The role of Bcl6 in mature cardiomyocytes
The 3rd Annual Scientific Meeting
P-I THE ROLE OF BCL6 IN MATURE CARDIOMYOCYTES
Yuji Matsudo'=,Takehiko Yoshida',Tetsuya Fukuda' Masahiko Hatano',Satoko Kojlma;Masafumi Arima' Takeshi Tokuhisa',Yoshiaki Masuda" ' Depatment of Oevelopemental Genetics, Chiba University Graduate School of Medicine 2Third department of internal medicine, Chiba University School of Medicine
The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle.The objective of this study was to examine the role of Bcl6 in cardiac myocytes. Method:We developed Bcl6-deficient(Bcl6 ~) mice and histlogically examined hearts from these mice. Results:Massive myocarditis with eosinophilic infiltration occured in Bcl6-~mice after 4 to 6 weeks of age.Since expression of the Bcl6 gene was induced in normal cardiac myocytes after two weeks of age and thereafter detected through adulthood,loss of Bcl6 in mature cardiac myocyte may be related with the induction of eosinophilic myocarditis.To examine effects of eosinophils from Bcl6 mice on normal hearts,bone marrow cells from Bcl6-J-mice were adoptively transferred into subtethally irradiated RAGl-deficient mice. Although massive eosinophilio infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Furthermore electron microscopic analysis of cardiac myocytes from Bcl6 Jmice revealed a spectrum of generative changes prior to eosinophilic infiltration. Conclution:Bcl6 is not essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation. We are examining the role of Bcl6 in Doxorubicin induced cardiomyopathy.
P-3 ~I-ADRENOCEPTOR-Gq SIGNALING PATHWAY IS UPFEEGULA'rED IN THE CARDIAC MYOCYTES OF TACHYPACING INDUCED FAILING HEART ASSOCIATED WITH I ~ S E D MYOFIBRILLAR Ca2+SENSITIVITY Nobuhim Suematsu, Shinji Satoh, Tomomi Ide, Shintam Kinugawa Hiroyuki Tsutsui, Akira Takeshita Research Institute of Angiocardiology, Kyushu University, =ukuoka 812-8582, Japan To investigate the c~-adrenoceptor-mediated regulation of myocardial contractility, we studied the effect of ~-adrenoceptor activation on the myofibdllar Ca 2* sensitivity using ~- toxin-skinned single cardiac cells isolated from tachypacing-induced failing heart in dogs. Resting sarcomere lengths were set at 2.02 pm (n=13) in normal and 1.99 pm (n=13) in heart failure (p=NS). Skinned cells were activated by varied [Ca 2+ ] (0.1 pM to 10 pM)in the absence or presence of 10 pM phenylephrine plus 100 pM GTP, and the developed isometric forces were measured. At baseline, maximum Ca 2. -activated force and myofibdllar Ca 2. sensitivity were comparable between normal and heart failure. While phenylephrine did not affect the maximum Ca 2÷ -activated force in two groups, it increased the myofibrillar Ca 2. sensitivity in heart failure (pCa ; from6.14 to 6.28, p<0.05) but not in normal (pCa ; from 6.14 to 6.21, p=NS). To determine the basis for the ~-adrenoceptor; mediated increase in the myofibrillar Ca 2+ sensitivity in heart failure, we quantified Gq protein expression in the left ventricle (LV) tissues. Western blot analysis showed a 1.6-fold increase in the Gq expression of the failing LV (n=6) compared with that of the normal LV (n=6, p<0.05). Our data indicate that ~-adrenoceptor-Gq signaling is augmented in the tachypacing-induced heart failure, resulting in increased myofibrillar Ca 2* sensitivity. This signaling pathway may work as the compensation mechanism for decreased myocardial contractility and also may partly contribute to the relaxation abnormality in heart failure.
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JHFS
P-2 The negative inotropic and lusitropic effect of beta3adrenoceptor stimulation on beating guinea pig heart Tetsuya Kitamura, Katsuya Onishi, Kaoru Dohi, Naoki Isaka, Takeshi Nakano The First Department of Internal Medicine, Mie University School of Medicine, Tsu 514-8507, Japan In the human failing ventdcular muscle, beta3adrenoceptor(AR) stimulation inhibits the contractile shortening. , However, cardiac performance and its mechanism of beta3AR in the whole heart are unclear. Therefore, we examined the effect of BRL37344(BRL), a beta3-AR agonist, on the left ventricular(LV) contractile performance and [Ca 2 ]~transients (with indo-1) in guinea pig Langendorff hearts (constant perfused pressure; 80 mmHg) under right ventricular pacing at 160 bpm. BRL significantly decreased LV developed pressure (control vs. BRL 10nM; 101+_.2vs. 81+_2 mmHg,p<0.05), peak +dp/dt (1755_+56 vs. 1408_+40 mmHg/sec, p<0.05), and peak dp/dt (826+~?.2 vs. 665 +_15 rnmHg/sec, p<0.05) in a dosedependent manner. These responses were accompanied by a relative decrease in systolic amplitude of [Ca2+]i transient. Pretreatment with 10nM nadolol, a selective beta l-/beta2-AR antagonist, preserved beta3 AR-induced negative inotropic and lusitropic responses. These effects were prevented in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 10 nM), a nitric oxide synthase (NOS) inhibitor. Conclusion: Beta3-AR stimulation elicits systolic and diastolic dysfunction associated with a decrease in [Ca2+], transient. These negative inotropic effects are likely to be rnediated through activation of a NOS pathway.
P-4 RELATIONSHIP BETWEEN pHi AND Ca;?.+ SENSmVFrY OF MYOFILAMENT8 IN RAT VENTRICULAR MYOCYTES Hajime Tarada, Hiroshi Satoh, Wei Uu, Hideharu Hayashi* Department of Intemal Medicine III, *Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3124, Japan The objectives of this study were to develop the method to measure Ca2+ transients (CAT), pHi and call length simultaneously, and to examine the relationship between phi and Ca2+ sensitivity of myofilaments in isolated ventdcular myocytes. Method: Microscopic fluorescent measurements were carried out in dual-loaded with indo-l/AM (10pM) and SNARF-1/AM (5pM) to measure [Ca2+]i and )Hi. Cell length was measured from the bright-field image. Intracallular acidosis or alkalosis were induced by propionate or NH4CI, respectively. Result: The method was validated since there was no interaction between each indicator. The amplitude of twitch cell shortening and CaT, and pHi in control myocytes were 5.0i-0.7 pm, 469±43 nM, and 7.27i-0.07, respectively. NH4CI induced an intracellular alkalosis (pHi: 7.66±O.08) and significantly increased the twitch cell shortening (10.5+1.3 pm) with no change in the amplitude of CaT. Propionate induced an intracellular acidosis (pHi:6.89-~-0.08) and significantly decreased the twitch call shortening (2.1±0.6 pm) with no change in the amplitude of CaT. The effects of pHi on the Ca2+ sensitivity were assessed with phase-plane analysis of call length and [Ca2+]i, Propionate or NH4CI shifted the trajectories to the right or to the left, respectively. The shift of the trajectories by intracellular alkalosis was larger than i that by acidosis. The ratios of % changes in cell shortening to CaT (CSt~aT) were decreased by pmpionate whereas NH4CI increased CS/CaT. To compare the relative influence of acidosis and alkalosis 'to the Ca2+ sensitivity, CS/CaT was divided by the change of pHi in each call (CS/CaT/ApHi:an index reflecting the change in the Ca2+ sensitivity by unit change in pHi). CS/CaT/ApHi in calls exposed to NH4CI was significantly larger than the index in calls exposed to propionate (8.1±1.5 vs 1.8~O,4). In conclusion, CaT, pHi and call length could be measured in an identical myocyte. The relationship between phi and Ca2+ sensitivity of myofilaments is not linear and the influence of pHi to the Ca2+ sensitivity is larger in alkaJesis.
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P-I THE ROLE OF BCL6 IN MATURE CARDIOMYOCYTES
Yuji Matsudo'=,Takehiko Yoshida',Tetsuya Fukuda' Masahiko Hatano',Satoko Kojlma;Masafumi Arima' Takeshi Tokuhisa',Yoshiaki Masuda" ' Depatment of Oevelopemental Genetics, Chiba University Graduate School of Medicine 2Third department of internal medicine, Chiba University School of Medicine
The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle.The objective of this study was to examine the role of Bcl6 in cardiac myocytes. Method:We developed Bcl6-deficient(Bcl6 ~) mice and histlogically examined hearts from these mice. Results:Massive myocarditis with eosinophilic infiltration occured in Bcl6-~mice after 4 to 6 weeks of age.Since expression of the Bcl6 gene was induced in normal cardiac myocytes after two weeks of age and thereafter detected through adulthood,loss of Bcl6 in mature cardiac myocyte may be related with the induction of eosinophilic myocarditis.To examine effects of eosinophils from Bcl6 mice on normal hearts,bone marrow cells from Bcl6-J-mice were adoptively transferred into subtethally irradiated RAGl-deficient mice. Although massive eosinophilio infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Furthermore electron microscopic analysis of cardiac myocytes from Bcl6 Jmice revealed a spectrum of generative changes prior to eosinophilic infiltration. Conclution:Bcl6 is not essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation. We are examining the role of Bcl6 in Doxorubicin induced cardiomyopathy.
P-3 ~I-ADRENOCEPTOR-Gq SIGNALING PATHWAY IS UPFEEGULA'rED IN THE CARDIAC MYOCYTES OF TACHYPACING INDUCED FAILING HEART ASSOCIATED WITH I ~ S E D MYOFIBRILLAR Ca2+SENSITIVITY Nobuhim Suematsu, Shinji Satoh, Tomomi Ide, Shintam Kinugawa Hiroyuki Tsutsui, Akira Takeshita Research Institute of Angiocardiology, Kyushu University, =ukuoka 812-8582, Japan To investigate the c~-adrenoceptor-mediated regulation of myocardial contractility, we studied the effect of ~-adrenoceptor activation on the myofibdllar Ca 2* sensitivity using ~- toxin-skinned single cardiac cells isolated from tachypacing-induced failing heart in dogs. Resting sarcomere lengths were set at 2.02 pm (n=13) in normal and 1.99 pm (n=13) in heart failure (p=NS). Skinned cells were activated by varied [Ca 2+ ] (0.1 pM to 10 pM)in the absence or presence of 10 pM phenylephrine plus 100 pM GTP, and the developed isometric forces were measured. At baseline, maximum Ca 2. -activated force and myofibdllar Ca 2. sensitivity were comparable between normal and heart failure. While phenylephrine did not affect the maximum Ca 2÷ -activated force in two groups, it increased the myofibrillar Ca 2. sensitivity in heart failure (pCa ; from6.14 to 6.28, p<0.05) but not in normal (pCa ; from 6.14 to 6.21, p=NS). To determine the basis for the ~-adrenoceptor; mediated increase in the myofibrillar Ca 2+ sensitivity in heart failure, we quantified Gq protein expression in the left ventricle (LV) tissues. Western blot analysis showed a 1.6-fold increase in the Gq expression of the failing LV (n=6) compared with that of the normal LV (n=6, p<0.05). Our data indicate that ~-adrenoceptor-Gq signaling is augmented in the tachypacing-induced heart failure, resulting in increased myofibrillar Ca 2* sensitivity. This signaling pathway may work as the compensation mechanism for decreased myocardial contractility and also may partly contribute to the relaxation abnormality in heart failure.
•
JHFS
P-2 The negative inotropic and lusitropic effect of beta3adrenoceptor stimulation on beating guinea pig heart Tetsuya Kitamura, Katsuya Onishi, Kaoru Dohi, Naoki Isaka, Takeshi Nakano The First Department of Internal Medicine, Mie University School of Medicine, Tsu 514-8507, Japan In the human failing ventdcular muscle, beta3adrenoceptor(AR) stimulation inhibits the contractile shortening. , However, cardiac performance and its mechanism of beta3AR in the whole heart are unclear. Therefore, we examined the effect of BRL37344(BRL), a beta3-AR agonist, on the left ventricular(LV) contractile performance and [Ca 2 ]~transients (with indo-1) in guinea pig Langendorff hearts (constant perfused pressure; 80 mmHg) under right ventricular pacing at 160 bpm. BRL significantly decreased LV developed pressure (control vs. BRL 10nM; 101+_.2vs. 81+_2 mmHg,p<0.05), peak +dp/dt (1755_+56 vs. 1408_+40 mmHg/sec, p<0.05), and peak dp/dt (826+~?.2 vs. 665 +_15 rnmHg/sec, p<0.05) in a dosedependent manner. These responses were accompanied by a relative decrease in systolic amplitude of [Ca2+]i transient. Pretreatment with 10nM nadolol, a selective beta l-/beta2-AR antagonist, preserved beta3 AR-induced negative inotropic and lusitropic responses. These effects were prevented in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 10 nM), a nitric oxide synthase (NOS) inhibitor. Conclusion: Beta3-AR stimulation elicits systolic and diastolic dysfunction associated with a decrease in [Ca2+], transient. These negative inotropic effects are likely to be rnediated through activation of a NOS pathway.
P-4 RELATIONSHIP BETWEEN pHi AND Ca;?.+ SENSmVFrY OF MYOFILAMENT8 IN RAT VENTRICULAR MYOCYTES Hajime Tarada, Hiroshi Satoh, Wei Uu, Hideharu Hayashi* Department of Intemal Medicine III, *Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3124, Japan The objectives of this study were to develop the method to measure Ca2+ transients (CAT), pHi and call length simultaneously, and to examine the relationship between phi and Ca2+ sensitivity of myofilaments in isolated ventdcular myocytes. Method: Microscopic fluorescent measurements were carried out in dual-loaded with indo-l/AM (10pM) and SNARF-1/AM (5pM) to measure [Ca2+]i and )Hi. Cell length was measured from the bright-field image. Intracallular acidosis or alkalosis were induced by propionate or NH4CI, respectively. Result: The method was validated since there was no interaction between each indicator. The amplitude of twitch cell shortening and CaT, and pHi in control myocytes were 5.0i-0.7 pm, 469±43 nM, and 7.27i-0.07, respectively. NH4CI induced an intracellular alkalosis (pHi: 7.66±O.08) and significantly increased the twitch cell shortening (10.5+1.3 pm) with no change in the amplitude of CaT. Propionate induced an intracellular acidosis (pHi:6.89-~-0.08) and significantly decreased the twitch call shortening (2.1±0.6 pm) with no change in the amplitude of CaT. The effects of pHi on the Ca2+ sensitivity were assessed with phase-plane analysis of call length and [Ca2+]i, Propionate or NH4CI shifted the trajectories to the right or to the left, respectively. The shift of the trajectories by intracellular alkalosis was larger than i that by acidosis. The ratios of % changes in cell shortening to CaT (CSt~aT) were decreased by pmpionate whereas NH4CI increased CS/CaT. To compare the relative influence of acidosis and alkalosis 'to the Ca2+ sensitivity, CS/CaT was divided by the change of pHi in each call (CS/CaT/ApHi:an index reflecting the change in the Ca2+ sensitivity by unit change in pHi). CS/CaT/ApHi in calls exposed to NH4CI was significantly larger than the index in calls exposed to propionate (8.1±1.5 vs 1.8~O,4). In conclusion, CaT, pHi and call length could be measured in an identical myocyte. The relationship between phi and Ca2+ sensitivity of myofilaments is not linear and the influence of pHi to the Ca2+ sensitivity is larger in alkaJesis.
53